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101.
102.
Seiji Abe Shigeo Nakabayashi Jun‐Ichiro Murayama Yoshihiro Sano Ken‐Ichi Ohno Masako Maeda Hidetoshi Arakawa 《Luminescence》2010,25(6):456-462
Nitric oxide (NO) is related to various physiological effects as well as to numerous diseases caused by accentuation of NO production. Measurement of NO in cells and tissues is difficult as NO readily reacts with other molecules; furthermore, its half‐life as a radical is fleeting. Currently, many NO pharmaceuticals are marketed as therapeutic agents for ischemic disease. Consequently, the identification of NO radicals and determination of generation rate from pharmaceuticals is very important when the effect of the medicinal supply is estimated. In this study, we developed a fluorometric assay for NO employing sesamol (3,4‐methylenedioxyphenol) as a fluorometric substrate. Sesamol is converted to a fluorescent derivative (ex. 365 nm, em. 447 nm), which is dimmer in the presence of NO. The detection limit of NO with this method is 400 fmol; moreover, NO generated from drugs can be measured. Copyright © 2009 John Wiley & Sons, Ltd. 相似文献
103.
Nagamine T Nakazato K Suzuki K Kusakabe T Sakai T Oikawa M Satoh T Kamiya T Arakawa K 《Biological trace element research》2007,117(1-3):115-126
This study undertook the analysis of tissue cadmium (Cd) distribution using in-air micro-particle-induced X-ray emission (PIXE)
and the examination of the involvement of metal ions in parenteral Cd toxicity. A mouse was injected intraperitoneally with
3 mg/kg body weight of CdCl2 thrice weekly. After 27 wk, the liver and kidney were excised and fixed in 10% formalin solution for 4 h and then embedded
in paraffin. Thin paraffin sections were used to analyze trace elements with in-air micro-PIXE and to examine metallothionein
protein and histological changes. Cd distribution was determined by micro-PIXE in the liver and renal cortex of the Cd-exposed
mouse, and the net Cd count was higher in the liver than in the renal cortex. The net iron (Fe) count was higher in the liver
of the Cd-exposed mouse compared to the control, and an opposite tendency was observed in the renal cortex. Wide cellular
Cd distribution was demonstrated in the liver and renal cortex of the chronic Cd-exposed mouse compared to the control. Metallothionein
staining was increased by chronic exposure to Cd both in the liver and kidney, and nephrotoxicity was more apparent than hepatotoxicity.
The modification of tissue Fe and calcium distribution by an intraperitoneal injection of Cd might be involved in Cd-induced
toxicity. 相似文献
104.
Kobayashi M Abiru N Arakawa T Fukushima K Zhou H Kawasaki E Yamasaki H Liu E Miao D Wong FS Eisenbarth GS Eguchi K 《Journal of immunology (Baltimore, Md. : 1950)》2007,179(4):2082-2088
Insulin peptide B:9-23 is a major autoantigen in type 1 diabetes that contains two distinct CD4 epitopes (B:9-16 and B:13-23). One of the two epitopes, B:13-23, overlaps with a CTL epitope (B:15-23). In this study, we report that the elimination of the CTL epitope from the B:9-23 peptide by amino acid substitution (with alanine) at positions B:16 and 19 (A16,19 altered peptide ligand) or truncation of the C-terminal amino acids from the peptide (B:9-21), neither of which stimulated the proliferation of insulin B:15-23 reactive CD8 T cells, provided significant intranasally induced suppression of diabetes when coadministered with a potent mucosal adjuvant cholera toxin (CT). Intranasal treatment with A16,19 resulted in the elimination of spontaneous insulin autoantibodies, significant inhibition of insulitis and remission from hyperglycemia, and prevented the progression to diabetes. Intranasal administration of native B:9-23/CT or B:11-23/CT resulted in a significant enhancement of insulin autoantibody expression and severity of insulitis and failed to prevent diabetes. Our present study indicates that elimination of the CTL epitope from the B:9-23 peptide was critically important for mucosally induced diabetes prevention. The A16,19 altered peptide ligand, but not other native insulin peptides, suppresses insulin autoantibodies associated with protection from and remission of diabetes. 相似文献
105.
Exposure of antibodies to low pH is often unavoidable for purification and viral clearance. The conformation and stability of two humanized monoclonal antibodies (hIgG4-A and -B) directed against different antigens and a mouse monoclonal antibody (mIgG1) in 0.1M citrate at acidic pH were studied using circular dichroism (CD), differential scanning calorimetry (DSC), and sedimentation velocity. Near- and far-UV CD spectra showed that exposure of these antibodies to pH 2.7-3.9 induced only limited conformational changes, although the changes were greater at the lower pH. However, the acid conformation is far from unfolded or so-called molten globule structure. Incubation of hIgG4-A at pH 2.7 and 3.5 at 4 degrees C over the course of 24 h caused little change in the near-UV CD spectra, indicating that the acid conformation is stable. Sedimentation velocity showed that the hIgG4-A is largely monomeric at pH 2.7 and 3.5 as well as at pH 6.0. No time-dependent changes in sedimentation profile occurred upon incubation at these low pHs, consistent with the conformational stability observed by CD. The sedimentation coefficient of the monomer at pH 2.7 or 3.5 again suggested that no gross conformational changes occur at these pHs. DSC analysis of the antibodies showed thermal unfolding at pH 2.7-3.9 as well as at pH 6.0, but with decreased melting temperatures at the lower pH. These results are consistent with the view that the antibodies undergo limited conformational change, and that incubation at 4 degrees C at low pH results in no time-dependent conformational changes. Titration of hIgG4-A from pH 3.5 to 6.0 resulted in recovery of native monomeric proteins whose CD and DSC profiles resembled those of the original sample. However, titration from pH 2.7 resulted in lower recovery of monomeric antibody, indicating that the greater conformational changes observed at this pH cannot be fully reversed to the native structure by a simple pH titration. 相似文献
106.
Shibayama K Wachino J Arakawa Y Saidijam M Rutherford NG Henderson PJ 《Molecular microbiology》2007,64(2):396-406
gamma-Glutamyltranspeptidase (GGT) is a periplasmic enzyme of Helicobacter pylori implicated in its pathogenesis towards mammalian cells. We have cloned and expressed the H. pylori strain 26695 recombinant GGT protein in Escherichia coli and purified it to homogeneity. The purified protein exhibited hydrolysis activity with very high affinities for glutamine and glutathione shown by apparent K(m) values lower than 1 muM. H. pylori cells were unable to take up extracellular glutamine and glutathione directly. Instead, these substances were hydrolysed to glutamate by the action of GGT outside the cells. The glutamate produced was then transported by a Na(+)-dependent reaction into H. pylori cells, where it was mainly incorporated into the TCA cycle and partially utilized as a substrate for glutamine synthesis. These observations show that one of the principle physiological functions of H. pylori GGT is to enable H. pylori cells to utilize extracellular glutamine and glutathione as a source of glutamate. As glutamine and glutathione are important nutrients for maintenance of healthy gastrointestinal tissue, their depletion by the GGT enzyme is hypothesized to account for the damaging of mammalian cells and the pathophysiology of H. pylori. 相似文献
107.
108.
Yoshihiro Shiomi Toshimasa Yamauchi Masato Iwabu Miki Okada-Iwabu Ryo Nakayama Yuki Orikawa Yoshichika Yoshioka Koichiro Tanaka Kohjiro Ueki Takashi Kadowaki 《The Journal of biological chemistry》2015,290(23):14567-14581
A novel peroxisome proliferator-activated receptor (PPAR) modulator, Z-551, having both PPARα agonistic and PPARγ antagonistic activities, has been developed for the treatment of obesity and obesity-related metabolic disorders. We examined the effects of Z-551 on obesity and the metabolic disorders in wild-type mice on the high-fat diet (HFD). In mice on the HFD, Z-551 significantly suppressed body weight gain and ameliorated insulin resistance and abnormal glucose and lipid metabolisms. Z-551 inhibited visceral fat mass gain and adipocyte hypertrophy, and reduced molecules involved in fatty acid uptake and synthesis, macrophage infiltration, and inflammation in adipose tissue. Z-551 increased molecules involved in fatty acid combustion, while reduced molecules associated with gluconeogenesis in the liver. Furthermore, Z-551 significantly reduced fasting plasma levels of glucose, triglyceride, free fatty acid, insulin, and leptin. To elucidate the significance of the PPAR combination, we examined the effects of Z-551 in PPARα-deficient mice and those of a synthetic PPARγ antagonist in wild-type mice on the HFD. Both drugs showed similar, but weaker effects on body weight, insulin resistance and specific events provoked in adipose tissue compared with those of Z-551 as described above, except for lack of effects on fasting plasma triglyceride and free fatty acid levels. These findings suggest that Z-551 ameliorates HFD-induced obesity, insulin resistance, and impairment of glucose and lipid metabolisms by PPARα agonistic and PPARγ antagonistic activities, and therefore, might be clinically useful for preventing or treating obesity and obesity-related metabolic disorders such as insulin resistance, type 2 diabetes, and dyslipidemia. 相似文献
109.
Kawai Y 《Bioscience, biotechnology, and biochemistry》2011,75(4):609-617
It has been suggested that polyphenol-rich diets decrease the risk of cardiovascular diseases. Although studies of the bioavailability of polyphenols, particularly their absorption and metabolism, have been reported recently, the tissue and cellular distributions underlying their biological mechanisms remain unknown. It is difficult to evaluate the specific localization of tissue and/or cellular polyphenols, because the method is limited to chromatography. To overcome these difficulties, we have developed anti-polyphenol antibodies to characterize immunohistochemically the localization of polyphenols and their metabolites in vivo. Two novel monoclonal antibodies were raised against quercetin and tea catechins, which represent flavonoid-type polyphenols distributed in foods and beverages, and are expected to exhibit anti-oxidative and anti-inflammatory activities in vivo. Using these antibodies, we identified activated macrophages as a specific target of these flavonoids during the development of atherosclerotic lesions. This review describes recent findings on the molecular actions of flavonoids that underly their anti-atherosclerotic activity in vivo. 相似文献
110.
Yang Y Kurokawa T Takahama Y Nindita Y Mochizuki S Arakawa K Endo S Kinashi H 《Bioscience, biotechnology, and biochemistry》2011,75(6):1147-1153
The 113,463-bp nucleotide sequence of the linear plasmid pSLA2-M of Streptomyces rochei 7434AN4 was determined. pSLA2-M had a 69.7% overall GC content, 352-bp terminal inverted repeats with 91% (321/352) identity at both ends, and 121 open reading frames. The rightmost 14.6-kb sequence was almost (14,550/14,555) identical to that of the coexisting 211-kb linear plasmid pSLA2-L. Adjacent to this homologous region an 11.8-kb CRISPR cluster was identified, which is known to function against phage infection in prokaryotes. This cluster region as well as another one containing two large membrane protein genes (orf78 and orf79) were flanked by direct repeats of 194 and 566 bp respectively. Hence the insertion of circular DNAs containing each cluster by homologous recombination was suggested. In addition, the orf71 encoded a Ku70/Ku80-like protein, known to function in the repair of double-strand DNA breaks in eukaryotes, but disruption of it did not affect the radiation sensitivity of the mutant. A pair of replication initiation genes (orf1-orf2) were identified at the extreme left end. Thus, pSLA2-M proved to be a composite linear plasmid characterized by self-defense genes and homology with pSLA2-L that might have been generated by multiple recombination events. 相似文献